AFM Investigations of Phase Separation in Supported Membranes of Binary Mixtures of POPC and an Eicosanyl-based Bisphosphocholine Bolalipid

Supported membranes prepared from binary mixtures of DOPC and the bolalipid C20BAS have been examined by atomic force microscopy (AFM). The supported membranes are phase separated to give a thicker DOPC-rich phase and a thinner bolalipid-rich phase for a range of lipid compositions. These results confirm an earlier prediction from mean field theory that phase separation is the thermodynamically stable state for membranes containing approximately equimolar C20BAS and double chain monopolar lipids with chain lengths exceeding 15 carbons. Hydrophobic mismatch between the monopolar lipid hydrocarbon chains and the membrane spanning bolalipid chains was suggested to provide the driving force for phase separation. The AFM results also show that the morphology of the mixed POPC:C20BAS supported membranes varies significantly with the conditions used to prepare the vesicles and supported membrane samples. The complex membrane morphologies observed are attributed to the interplay of several factors, including a compositionally heterogeneous vesicle population, exchange of lipid between the vesicle solution and solid substrate during formation of the supported membrane, and slow equilibration of domains due to pinning of the lipids to the solid support.Peer reviewed: YesNRC publication: N

Phase Separation in Binary Mixtures of Bipolar and Monopolar Lipid Dispersions Revealed by 2H NMR Spectroscopy, Small Angle X-Ray Scattering, and Molecular Theory

Binary mixtures of C20BAS and POPC membranes were studied by solid-state 2H NMR spectroscopy and small angle x-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Three specifically deuterated C20BAS derivatives—[1′,1′,20′,20′-2H4]C20BAS, [2′,2′,19′,19′-2H4]C20BAS, and [10′,11′-2H2]C20BAS—combined with protiated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as membranes containing POPC-d31 and fully protiated bolalipid, were used in NMR experiments to obtain structural information for the mixtures. The 2H NMR spectra of [10′,11′-2H2]C20BAS/POPC membrane dispersions reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations (100, 90, and 70 mol %). At ≤50 mol % C20BAS, smaller quadrupolar couplings appear in the spectra, indicating the presence of U-shaped conformers. The proportion of U-shaped bolalipids increases as the amount of POPC in the membrane increases; however, the transmembrane component remains the dominant bolalipid conformation in the membrane even at 45°C and 10 mol % C20BAS, where it accounts for ∼50% of the bolalipid population. The large fraction of C20BAS transmembrane conformers, regardless of the C20BAS/POPC ratio, together with the findings from molecular mean-field theory calculations, suggests the coexistence of phase-separated bolalipid-rich domains and POPC-rich domains. A single lamellar repeat distance was observed in SAXS experiments corresponding to the average repeat spacing expected for C20BAS- and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples that arise from POPC-C20BAS hydrophobic mismatch